Our goal is to find out how the virus "negotiates" the entry of its genetic material into the cell. Cells are surrounded by cell membranes which impose insurmountable barriers for passage of undesirable molecules and particles into the cell. Viruses are enveloped by similar membranes. The virus has developed strategies to overcome these insurmountable barriers by designing envelope glycoproteins which catalyse the fusion of viral and cellular membranes. We are specifically studying the mode of action of the envelope glycoproteins of Human Immunodeficiency Virus (gp120-gp41), Murine Leukemia Virus, Vesicular Stomatitis Virus, Paramyxovirus, and Influenza virus. Using quantitative fluorescence videomicroscopy we have monitored kinetics of fusion between cell pairs consisting of a single influenza hemagglutinin (HA)-expressing cell and a single erythrocyte (RBC) which had been labeled with both a fluorescent lipid (DiI) in the membrane and a fluorescent solute (calcein) in the aqueous space. Initial fusion pore opening between the RBC and HA-expressing cell produced a change in RBC membrane potential which was monitored by a decreases in DiI fluorescence. This event was followed by two distinct stages of fusion pore dilation: the flux of fluorescent lipid and the flux of a large aqueous fluorescent dye. We have analyzed the kinetics of events which occur as a result of transitions between the various stages of fusion pore opening. We are developing similar data on HIV-1 envelope glycoprotein-mediated membrane fusion. Recently we have been able to show that glycolipids might be important players in HIV-1 entry. Knowledge of the mechanism of viral fusion pore expansion will have wide implications for the development of anti-viral strategies. Moreover, the knowledge we gained about the parameters and "design principles" derived from studies with HA and gp120-gp41 provide a conceptual basis for constructing chimeric viral envelope proteins which may be used as components of targeted systems negotiating entry of therapeutic agents into cells. This is of particular interest in the area of gene therapy.